On Oct 22, 1:59 pm, Han Xu <h... from cns.nyu.edu> wrote:
One thing I
> doubt is the internal pipette solution with an osmolarity of 271
> mOsm, which is low compared with the value mentioned in literature in
> the range of 290-310 mOsm. Is there anybody has experience on this
> problem? I will be really appreciated if somebody can give me any
> suggestions.
>> Best,
> Han
Hi Han,
First, I think it would be better if you adjusted your internal and
external osmolarity. We use about 310-315 for internal and about
320-325 for external, both adjusted upward with sucrose. Emprically,
having slightly higher external osmolarity tends to give longer, more
stable recordings.
Second, it is often not clear whether rising series resistance is
becase of a) the patch not being sufficiently ruptured, thus falling
back into the tip of the pipette, or b) internal organelles in the
cell, e.g., ER or nucleus, rising up and plugging the tip of the
pipette. The former can sometimes be fixed by adding negative pressure
(sucking) to the pipette, and the latter can sometimes be fixed by
positive pressure (blowing), but the most likely thing to happen if
you try to fix it is that you lose the recording. I find that once the
problem shows up, there's usually little that can be done. So the
trick is to get a stable recording that won't eventually have series
resistance problems in the first place.
Here is my personal strategy:
a) adjust osmolarities, as above.
b) we use pipettes with pretty low resistance to start with, like
1.5-3 MOhm. I think this helps.
c) when you first release the positive pressure to make a seal, the
cell will naturally rise up and push against your pipette. After
breaking in, this pushing may turn into plugging. So to avoid this,
AFTER I release the pressure and make a seal, but BEFORE breaking in,
I withdraw the pipette by a few microns. This almost never causes loss
of the seal, but it puts additional space between your pipette and the
ER and nucleus, so it can help avoid plugging.
d) When I do finally break in, I watch the capacitive transients very
closely to make sure that they are big, sharp and STABLE. Many times,
right after the initial break, the transients will get big quickly,
but then drift down to slightly smaller amplitudes over the next
several seconds. I think this is a sign that there's some bits of
patch that are trying to fall back into the tip, rather than sticking
to the sides of the glass. If I see this, I immediately maintain
gentle suction until they grow back up again. Then let the suction off
and watch to see that they stay that way for at least 10-20 seconds or
more, without shrinking again.
I find that if I establish these conditions at the very beginning, the
recording tends to stay stable much longer (i.e., a couple of hours,
with essentially no further change in Rs, in the best cases).
Matt
PS - also, make sure that your manipulators are not drifting at all,
and that your harp or grid is really holding the slice perfectly
still. If manipulators or slice are wobbling, Rs problems will ensue.